Experimental Gerontology, Vol. 24, pp. 501-513, 1989 0531-5565/89 $3.00 + .00 Printed in the USA. All fights reserved. Copyright © 1989 Pergamon Press pie

INCREASE IN ABUNDANCE OF A TRANSCRIPT HYBRIDIZING TO ELONGATION FACTOR I ALPHA DURING CELLULAR

SENESCENCE AND QUIESCENCE

TONY GIORDANO, 1 DON KLEINSEK 2 and DOUGLAS N. FOSTER 3

~Laboratory of Molecular Biology, National Institutes of Health, Building 37, Room 2El0, Bethesda, Maryland 20892, 2Baylor College of Medicine, One Baylor Plaza, Texas Medical Center, Houston, Texas 77030 and

3Department of Poultry Science, The Ohio State University, Ohio Agriculture Research and Development Center, Wooster, Ohio 44691

Abstract - - We have isolated a senescence-specific clone (pSEN) from a cDNA library constructed from late passage WI-38 human diploid fibroblast that accounts for approxi- mately 1% of the recombinants. Nucleotide sequence analysis of the partial cDNA clone has led to the identification of pSEN as elongation factor I alpha. Northern analysis of poly(A) + RNA from various intermediate population doubling levels shows that a 2.2 kb transcript hybridizes to pSEN but is expressed prior to PDL-40 at very low levels. This transcript begins to accumulate at PDL-40 and is induced approximately 50-fold just prior to senescence. Furthermore, this transcript was shown to be specific to G o of the cell cycle whereas a second, lower molecular weight transcript (1.6 kb) was observed during S phase (Giordano and Foster, unpublished data). The 2.2 kb transcript is also detected in neonatal foreskin cells but very little increase in abundance is observed between early and late passage cells. Sucrose gradient fractionation of RNA from late passage WI-38 cells suggests that the lower molecular weight transcript is associated with the polysome fraction while the 2.2 kb transcript sediments with the nonpolysomal fraction. Thus, the possibility exists that the 1.6 kb transcript is derived from the 2.2 kb transcript.

INTRODUCTION

Ctaoror, t ~ NORMAL human fibroblasts undergo a finite number of population doublings that are inversely correlated to the age of the donor (Hayflick, 1965). In vitro cellular senescence has been utilized as a model system for in vivo aging. However, the mechanisms leading up to senescence are not yet well defined. Lumpkin et al. (1986) reported an antiproliferative po ly (A)+ RNA accounting for between 0.1 and 1% of the po ly (A)+ RNA in senescent cells,

Salaries and research support provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University, manuscript number 309-88. Correspondence to: T. Giordano.

501

502 T. GIORDANO et aL

but it has not yet been identified. We have attempted to identify this, or other highly abundant sequences from senescent cells, by differentially screening a cDNA library derived from late passage cells. One such clone was isolated and identified as elongation factor 1 alpha (EF-1 alpha) by nucleotide sequence analysis.

Elongation factor 1 alpha (EF-1 alpha) is a protein which functions in translation by binding amino-acyl tRNA to the acceptor site of ribosomes. Cavallius et al. (1986) have shown that the content and activity of the protein decreases as human cells near senescence. Bolla and Brot (1975) reported that during nematode aging there appears to be a conversion of the EF-1 alpha protein from a higher to lower molecular weight form with a concomitant increase in the number of inactive or partially active molecules. Likewise, EF-1 activity has been shown to decline during the life span of rats (Moldave et al., 1979). We report here the isolation of a clone for EF-1 alpha that exhibited increased hybridization to a cDNA probe derived from late passage cells. The apparent paradox between the increase in the EF-1 alpha transcript we observe and the decrease in the content and activity of the protein is discussed.

MATERIALS AND METHODS

Cell culture

WI-38 cells (human fetal lung fibroblasts with a normal karyotype) were obtained from the Coriell Institute for Medical Research, Camden, NJ (repository number AG68140) at popula- tion doubling level (PDL) 14. Cells were cultured at 37 ° C, 5% CO 2 in McCoy's 5A medium containing 10% fetal bovine serum. Cultures used in cell cycle analysis were grown to subconfluence and then changed after 5 days to McCoy's 5A medium containing 0.5% fetal bovine serum for 10 days. The cells were stimulated to enter the cell cycle by addition of medium containing 20% fetal bovine serum.

RNA isolation

Thirty minutes prior to harvest 1 i~g/ml cycloheximide was added to the cultures to stabilize mRNA. Total cellular RNA was isolated from subconfluent cultures 64 to 72 h after plating or refeeding using the methods of Ullrich et al. (1977). Poly(A)+ RNA was isolated from total RNA by double passage over an oligo-d(T) cellulose column. For the cell cycle analysis, total RNA was harvested at 0, 4.5, 9.0, 13.5, 18.0, and 22.5 h after serum stimulation. The degree of cell cycle synchronization was determined by 3H-thymidine labelling followed by autorad- iography. Total RNA was isolated for polysome gradient analysis 10 h after serum stimulation.

Library construction and screening

A cDNA library was constructed in the bacteriophage vector lambda gtl0 using commercial synthesis and cloning kits (Amersham, Arlington Heights, IL) with poly(A)+ RNA obtained from cells which had completed more than 95% of their replicative potential. Replicate nitrocellulose filters were differentially hybridized with random-primed (32p) labelled cDNA (Feinberg and Vogelstein, 1983) derived from subconfluent early and late passage cells at 43°C for 24 h in buffer containing nonfat dry milk (Johnson et al., 1984). Filters were washed in 0.2X

INCREASE IN TRANSCRIF ABUNDANCE 503

10 20 30

50

48

46

44

42

40

38

36

34

32

30

28

26

24

22

20

18

16

14 i

130 46 50 6b 7b 8b 9b zo~ lio z~o Number Days In C u l t u r e

FIO. 1. Growth curve of WI-38 cells ineulture. Cells were grown in McCoy's 5A and 10% fetal bovine serum and passaged prior to confluence.

SSC, 0.1% SDS for 1 h at 55°C. A total of 20 000 eDNA clones were screened. A clone which exhibited increased hybridization to the late passage probe was subcloned into the EcoR1 site of pUC 119 and denoted as pSEN. The pUC 119 was a generous gift from M. McMullen (Ohio State University) and used because of its utility as a single-strand sequencing vector.

RNA Blot hybridization

RNA (1 ttg of poly(A)+ or 20 ttg of total RNA per sample) was denatured in 50% formamide at 65 °C for 15 min and then separated by electrophoresis through a 1.5% agarose/formaldehyde gel in MOPs buffer (Maniatis, et al. 1982). After denaturation and neutralization of the gel, the RNA was transferred to Zeta-probe membranes (Bio-Rad, Richmond, CA), baked, prehybrid-

504 T. GIORDANO et al.

Poly A+ ~ from pdl:

- - 2 . 2 k b

FIG. 2. Northern analysis of RNA from early- mid- and late-passage cells. One 0.g/lane of poly(A)+ RNA was loaded onto a denaturing agarose gel, separated by electrophoresis, transferred to nitrocellulose, and probed with (32p) labelled pSEN.

ized and hybridized for 24 h at 43 °C to (32p) labelled pSEN in buffer containing diethylpyro- carbonate treated nonfat dry milk (Siegel and Bresnich, 1986). The filters were washed in 0.2 × SSC, 0.1% SDS for 60 min at 65°C, then exposed to Kodak XAR-5 x-ray film for 1 h using a Lightning Plus intensifying screen.

Polysome gradient

Monolayers of late passage WI-38 cells (grown as described for cell cycle analysis) were scraped in 1 ml of 0.01 M NaCI, 3 mM MgC12, 0.01 M Tris-HC1, and 0.5% Nonidet P-40, then vortexed and centrifuged at 800 × g for 2 min at 4 °C to remove nuclei. Heparin was added to a final concentration of 1.5 mg/ml to inhibit nuclease activity and the sample was layered on a 15 to 40% sucrose gradient in lysis buffer minus Nonidet P-40. The gradients were centrifuged at 32 500 rpm in a SW-41 rotor for 2 h at 4°C (Geyer et al., 1982). Samples (500 p,l) were collected from the top of the gradient, ethanol precipitated, and analyzed by RNA blot- hybridization to a (32p) labelled pSEN probe as described above.

RESULTS

WI-38 cells exhibit a biphasic growth curve (Fig. 1) consisting of an initial exponential growth period in which the population doubles every 20 to 24 h, referred to as phase II, followed by a period of declining replicative activity, noted as phase III. A cDNA library was constructed from RNA obtained from cells very near the end of phase III and was differentially screened

INCREASE IN TRANSCRIPT ABUNDANCE 505

PDL-32

WI-38 Cells

PDL-45

FIc. 3. Photomicrograph of early- and late-passage WI-38 cells. The photo on the left is of a culture at PDL-32, the photo to the right is of cells at PDL-45. Note the increased size and number of appendages in the late passage cells.

with probes derived from late phase III (98% replicative life span completed (RLC)) and early phase II cells (42% RLC). From a primary screening of approximately 20 000 recombinants, a clone which exhibited a much greater hybridization intensity to the phase III probe was plaque purified and subcloned into pUC 119. This clone, referred to as pSEN, accounts for approximately 1% of the recombinants in the late passage library. The pSEN clone isolated in this study has been identified as human elongation factor 1 alpha (EF-1 alpha) by nucleotide sequence analysis (Giordano and Foster, submitted).

An analysis of the poly(A)+ RNA from subconfluent early-, mid- and late-passage cells confirmed that the pSEN transcript accumulates as cells near senescence. The northern blot in Fig. 2 shows a low level of hybridization of pSEN to a single 2.2 kb transcript from RNA isolated from early or mid-passage cells but high levels of hybridization to the 2.2 kb transcript with RNA from late passage cells. We have shown that this increase in hybridization occurs as cells enter phase III growth (Giordano and Foster, submitted). Concomitant with the accumulation of the transcript was a shift in the growth kinetics and change in cellular morphology. Cells exhibit an increase in cellular volume leading to a gradual 10-fold decrease in saturation density at the time of senescence (Fig. 3).

506 T. G1ORDANO et al.

A B

- 2 .2kb

Fic. 4. Effects of cycloheximide on abundance level of 2.2 kb transcript. WI-38 cellular RNA was harvested by scraping cells in the guanidium isothiocyanate at 4°C with (lane A) or without (lane B) 1 Izg/ml of cycloheximide added prior to harvest. Cultures used in this experiment were at PDL-47.

In the present study, cells were grown in the presence of 1 i~g/ml of cycloheximide for 30 min. prior to harvest to stabilize mRNA (Slobin and Jordan, 1984). Preliminary studies showed no difference in abundance levels of the 2.2 kb pSEN hybridizing transcript when isolated in the absence or presence of cycloheximide (Fig. 4).

Both early and late passage cells were serum arrested and subsequently stimulated with 20% fetal bovine serum to enter the cell cycle in an effort to assess at what stage of the cell cycle pSEN accumulated. Following 10 days in 0.5% serum, less than 1% of both early and late passage cells were synthesizing DNA (Table 1). No synthesis was detected until 13.5 h poststimulation when both cell populations began to incorporate 3H-thymidine in their DNA with maximal incorporation occurring at 18 h. At 18 h poststimulation 81% of the cells from the early-passage population had incorporated 3H-thymidine into their DNA. In contrast, only 35% of the cells from the late-passage population showed incorporation.

Total cellular RNA was immobilized on nitrocellulose with a slot blot apparatus for each population at each time-point and hybridized with pSEN to determine at what point accumu- lation of the transcript occurred (Fig. 5). In late-passage cells the transcript hybridized to pSEN maximally at t = 0 h and gradually decreased until t = 22.5 h when a slight increase in accumulation (relative to t = 18 h) was observed. Conversely, early-passage cells exhibited only a slight accumulation at t = 0 while maximal accumulation occurred at t = 18 h. To determine if these differences were due to a change in regulatory control of the single 2.2 kb transcript detected previously or if multiple related transcripts were hybridizing, a northern blot

INCREASE IN TRANSCRIPT ABUNDANCE

TABLE 1. P~RCE~rrAOE OF WI-38 CELLS ~COP.r'OP.AT~6 3H-TdR Tm~OUGHotrr CELL CYCLE

507

Hours into Cell Cycle After 20% Serum Stimulation Early (pdl 28) Late (pdl 49)

0 0.9% (8/840) 0.7% (5/690) 4.5 0.7% (8/1200) 0.6% (4/620) 9.0 0.6% (5/840) 0.4% (2/526)

13.5 27% (66/246) 19% (75/394) 18.0 82% (251 307) 35% (90/254) 22.5 75% (268/358) 26% (79/300)

Early- and late-passage cells were serum starved for 10 days at 0.5% fetal bovine serum then stimulated to enter the cell cycle with 20% serum. Cultures were labelled with 3H-TdR for 1 h prior to fixing then processed autoradiography. The number of labelled/unlabeled nuclei were determined.

of total cellular RNA was hybridized with pSEN (Giordano and Foster, submitted). Indeed, two transcripts of different sizes were observed: the 2.2 kb transcript was detected in late-passage cells and a lower molecular weight, 1.6 kb, transcript in early-passage cells. Upon close inspection, the 1.6 kb transcript was also observed in t -- 9 h RNA from late passage cells.

A 2.2 kb transcript was also observed when RNA from confluent early- and late-passage neonatal foreskin cells was subjected to northern blot analysis and hybridized to pSEN but with no difference in levels of hybridization (Fig. 6A). When a second northern blot of neonatal foreskin RNA was analyzed, this time with subconfluent early passage cells included, only slight differences in hybridization were observed (Fig. 6B). Again, the 2.2 kb transcript was the transcript detected in all instances.

Cultured Syrian hamster fibroblasts also exhibit a limited in vitro life span that is inversely related to the age of the donor (Bruce et al . , 1986). Preliminary studies using the pSEN probe to analyze total cellular RNA isolated from postconfluent Syrian hamster cells indicate a single transcript of approximately 1.8 kb, the abundance of which does not appear to change between early, mid, or late passage (Bruce, personal communication).

Rao and Slobin (1987) showed that when quiescent Friend erythroleukemia cells are treated with fresh serum, there is a shift of association of EF-1 alpha mRNA to heavy polysomes. We found that at 10 h poststimulation of late-passage WI-38 cells, when both the 2.2 and 1.6 kb transcripts are detectable, the 2.2 kb transcript is associated primarily with the nonpolysomal fraction (upper band, lanes 4-6) of a sucrose gradient while the 1.6 kb transcript is detected in the polysome fraction (lower band, lanes 12-18; Fig. 7).

DISCUSSION

The molecular mechanisms involved in cellular senescence are poorly defined. Many proteins have been identified which change their content in cells as they senesce (for review see Makrides, 1983); however, none have been implicated in preventing the cell from replicating. Pereira-Smith et al. (1985) and Stein and Atkins (1986) have shown that a membrane-associated

508 T. GIORDANO et al.

Total cellular RNA slotted

pdl 28.56

pdl 49.17

1.25ug 2.5ug

0

'~.5

9 13.5 18

22.5

0 4.5

9 13.5

18

22.5

Hrs into cell cycle a f t e r 20% serum stimulation

Fio. 5. Slot blot analysis of cell cycle specific RNA. Total cellular RNA from PDL-29 and 49 from specified time points following serum stimulation, was loaded onto a nitrocellulose with a slot blot apparatus and hybridized to (32p) labelled pSEN. Following hybridization the blot was washed and processed for autoradiography as described in the text.

protein appears to have antiproliferative activity but it has yet been identified. Cell fusion studies also support the argument that a protein is involved in the inhibition of replication (Burmer e t a l . , 1982; Drescher-Lincoln and Smith, 1984). Indeed, microinjected poly(A)+ RNA from nondividing rat liver cells (Lumpkin e t a l . , 1985) or senescent fibroblasts (Lumpkin e t a l . , 1986) inhibits replication of early passage fibroblasts. Thus, we attempted to isolate, identify and characterize a poly(A)+ derived cDNA from late-passage WI-38 cells by differentially screening a late-passage cDNA library.

The differential screening of the late-passage library with cDNA probes derived from early- and late-passage cells resulted in the isolation of a number of clones which exhibited different levels of hybridization to the probes. The clone which showed the greatest increase in hybridization to the late passage probe was subcloned and identified as EF- 1 alpha by nucleotide sequence analysis. This clone hybrized to approximately 1% of the recombinants in the late passage library.

WI-38 cells exhibit a biphasic growth curve consisting of expontential division followed by a gradual lengthening of the cell cycle. In addition to the morphological changes which occur as cells enter phase III growth, changes in excision repair (Mattern and Cerutti, 1975), EF-1 alpha content and activity (Cavallius e t a l . , 1986), Fibronectin (Oka, 1985), and glycolysis

INCREASE IN TRANSCRIFr ABUNI)ANCE 509

6B l 2 3

6

!

6A l 2

2.2kb

Fro. 6. Northern analysis of neonatal foreskin cellular RNA. Ten p,g/lane of total RNA was electrophoreseM, transferred to nitrocellulose, and hybridized to pSEN. 6A) RNA from confluent early and late passage cells. 6B) Lane l contains RNA from confluent, early; lane 2 from confluent, late; and lane 3 from subconfluent, early-passage cells.

(Bittles and Harper, 1984) have also been reported. We find that at this transition period cells begin accumulating a 2.2 kb transcript which hybridizes to EF-1 alpha. The increase in the abundance of the 2.2 kb transcript appears to be inversely correlated to the content and activity of the protein reported by Cavallius et al. (1986).

Orgel (1963) has proposed that during aging an increase in transcriptional and/or translational errors leads to an accumulation of aberrant proteins resulting in the loss of function. In fact, an accumulation of inactive or partially active EF-1 alpha macromolecules occurs during aging in Turbatrix aceti (Bolla and Brot, 1975). Thus, it is possible that during senescence the efficiency of translation of the EF-1 alpha transcript declines such that activity and content decline; comparable findings have been reported in this issue for other proteins. (Seshardri and Campisi, p. 515; Chen et al. p. 523). The increase in transcript may result from nonutilization of the transcript, a decrease in proteins responsible for turnover of transcripts, or feedback resulting in increased transcription to compensate for the decreased production of the EF-1 alpha protein.

Alternatively, the 2.2 kb transcript may not be directly translated. We observe that the 2.2 kb transcript is associated with the Go phase of the cell cycle for WI-38 and furthermore is found in the nonpolysomal fraction of a sucrose gradient. Slobin and Jordan (1984) report that EF-1 alpha mRNA is associated with a large percentage of Friend erythroleukemia cellular ribonucleoprotein particles (RNP). Furthermore, they find that in quiescent cells 60% of EF-1 alpha mRNA is found in RNP whereas, in serum stimulated cells 90% of EF-1 alpha mRNA is associated with polysomes (Rao and Slobin, 1987). They conclude that mRNA may be shifted from polysomes to RNPs and is then degraded.

We would like to propose an alternative model, that is, a 2.2 kb EF-1 alpha transcript is stored in RNPs, transferred to polysomes in an event involving processing to a 1.6 kb rnRNA species and then it is translated and degraded. During quiescence the 2.2 kb transcript would increase

510 T. GIORDANO et al.

in abundance due to increased stability of EF-1 alpha mRNA ( t l / 2 ---- 24 h in quiescent cells; t~/2

= 1 h in proliferating cells) in quiescent cells (Rao and Slobin, 1988). The 3' end of the transcript contains the sequence CACAG which is known to interact with the small nuclear ribonucleoprotein U4 (Opdenakker et al . , 1987) providing a means for stable association with RNPs. Upon serum induction, we propose that the transcript is processed to a 1.6 kb mRNA and becomes associated with polysomes where it is translated. This processing may involve cleavage of the 3' tail which could decrease stability of the transcript.

Active EF-1 alpha mRNA has recently been shown to be associated with S phase of the cell cycle (Lu and Werner, 1988) which is consistent with our observation of the 1.6 kb transcript being associated with the polysome fraction and induced by serum. De novo transcription of the 1.6 kb transcript is not believed to be occurring since work in other labs has demonstrated an increase in EF-1 alpha protein during serum stimulation in the presence of actinomyocin D (Thomas and Thomas, 1986). If the above model turns out to be correct, senescence may be the result of inefficient processing of the 2.2 kb transcript to translatable mRNA.

The inability to detect the 2.2 kb transcript in Syrian hamster cells can not be explained by lack of homology because a 1.8 kb transcript was detected. One possibility is that EF-1 alpha transcripts do not accumulate in RNPs in this cell line or are processed rapidly to lower molecular weight mRNA in all growth stages. A second possibility is that the 2.2 kb transcript is homologous to, but different from EF-1 alpha, and that the homology between the related transcript to Syrian hamster cells and EF-1 alpha in human cells is not sufficient for hybridization.

Human EF-1 alpha is part of a polymorphic and conserved multigene family (Opdenakker et

al . , 1987). In Drosophila, two genes encode two cytoplasmic forms of EF-1 alpha, both of which are functional (Hovemann et al . , 1988). A similar situation exists in Saccharomyces,

which also encodes two functional mRNAs (Cottrelle et al., 1985). Thus, the human multigene family may be transcriptionally active for two or more genes resulting in the detection of two transcripts. Recently, Moutsatsos et al. (1988) has shown that statin, a protein found in quiescent and senescent cells (Wang, 1985a, b), has considerable homology to EF-1 alpha. The 2.2 kb transcript we detect may be a statin or statin-like molecule.

If the second model is correct, it would appear that transcriptional regulation of the gene family is tightly coupled to the growth state of the cells. However, the function of the EF-1 alpha-like molecule is not known. It may simply be that both forms bind amino acyl tRNA transferring it to the acceptor site on the ribosome with one form functioning during quiescence, including senescence, while the other form is active during proliferation. Alternatively, the 2.2 kb transcript may code for a protein with entirely different function perhaps interfering with the translational machinery. Work is now in progress to determine the role of the 2.2 kb transcript in senescence and quiescence.

SUMMARY

A cDNA library was constructed from late passage WI-38 cells and differentially screened with cDNA probes derived from early and late passage cells. A clone was isolated which showed increased hybridization to the late-passage probe and identified as elongation factor 1 alpha by nucleotide sequence analysis.

The clone hybridized to a 2.2 kb transcript with increased hybridization occurring during phase III cellular growth and during quiescence. Additionally, a 1.6 kb transcript was observed during the S phase of the cell cycle. The 1.6 kb transcript appears to sediment with the polysome

1

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5 10 15 20

511

Fro. 7. Northern analysis of total RNA from PDL-49 cells after separation on a sucrose gradient. Cytoplasmic RNA was isolated from 10-h serum-induced, late-passage WI-38 cells and run through a 15 to 40% sucrose gradient. Twenty samples (500 p,l each) were then collected from the top (nonpolysomal) to the bottom (polysomal) of the gradient, precipitated in the presence of tRNA carrier, resuspended and loaded in lanes 1 through 20, respectively. After transfer to Zeta-probe, the filter was probed with (32p) labelled pSEN. Following hybridization, the filter was washed at 43°C in 0.2X SSC, 0.1% SDS, dryed and exposed to x-ray film. The upper band migrates at 2.2 kb and the lower band at 1.6 kb.

fraction on a sucrose gradient while the 2.2 kb transcript appears to be associated with the nonpolysomal fraction.

We propose two models for the occurrence of multiple transcripts. The first model suggests that a 2.2 kb transcript is associated and stored on RNPs during quiescence and is processed, transported to polysomes, and translated during S phase as a 1.6 kb transcript. The second model proposes the existence of two genes, each regulated at different stages of the cell cycle, each transcribing homologous transcripts. Current studies are aimed at determining which (if either) model is correct.

Acknowledgments -- We would like to thank Mike McMullen (Ohio State University, Dept. of Agronomy) for his generous gift of the pUC 119 plasmid. We are grateful for the suggestions and criticisms of this manuscript made by Sarah Brace and Keith Cook.

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